Small nucleic acid quantification using split cycle amplification

ABSTRACT

Methods of detecting or quantifying short RNA or DNA molecules using split cycle amplification are provided.

CROSS-REFERENCES TO RELATED APPLICATIONS

This application is a divisional of U.S. patent application Ser. No.15/046,297, filed Feb. 17, 2016, which claims priority to U.S.Provisional Application No. 62/117,381, filed Feb. 17, 2015, thecontents of each of which are hereby incorporated by reference in theirentireties for all purposes.

REFERENCE TO A “SEQUENCE LISTING,” A TABLE, OR A COMPUTER PROGRAMLISTING APPENDIX SUBMITTED AS AN ASCII TEXT FILE

The Sequence Listing written in file 094868-1117396_ST25.TXT, created onDec. 3, 2018, 8,448 bytes, machine format IBM-PC, MS-Windows operatingsystem, is hereby incorporated by reference in its entirety for allpurposes.

BACKGROUND OF THE INVENTION

A number of types of non-coding short RNAs occur in cells. Examples ofsuch RNAs include but are not limited to miRNA, snoRNA, piRNA, orlncRNA. Other types of RNA include, for example, mRNA. Shorter RNAs inparticular can present difficulties for amplification because of thesequences do not present a long enough sequence to hybridize and amplifyusing standard primers and methods.

Conventional PCR amplification and detection of nucleic acid targetsthat are shorter than 30 nucleotides long can be difficult. PCRamplification requires that the primers used for amplification anneal tothe target DNA or RNA at a temperature that is within the range of thepolymerase used in the reaction. Since PCR requires the reaction to beheated to 90° C. or higher during each cycle in order to melt theduplexes of nucleic acid materials, the polymerase must be heat stable.This is achieved by utilizing polymerases from thermophiles and resultsin an enzyme that functions best at temperatures centering on 60° C. butcan range between 40° C. and 75° C. As temperatures get near the lowerand higher ranges of the range of temperature the polymerase is lessefficient resulting in an ideal primer melting temperature between 50°C. and 65° C. This requirement results in primers that are between about15 and 30 nucleotides in length. Therefore the minimum amplicon ortarget length is about 30-60 nucleotides for DNA binding dye detectionand 45-90 nucleotides for Taqman probe-based detection. These lengthsalso depend on the GC content of the target so that, for example, a veryAT-rich target would require a much longer amplicon length than aGC-rich one.

BRIEF SUMMARY OF THE INVENTION

In some aspects, methods of quantitating an amount of a target DNAtemplate in a sample are provided. In some embodiments, the methodcomprises:

a) forming a plurality of mixture partitions, wherein the mixturepartitions comprise:

i) the target DNA template;

ii) a thermostable DNA dependent DNA polymerase; and

iii) a forward and a reverse amplification primer, wherein

the amplification primers comprise a 3′ hybridization region thathybridizes to the target DNA template and primes template directedextension of the primer in the presence of the DNA dependent DNApolymerase;the forward or the reverse amplification primer optionally furthercomprises a 5′ tail region that is not complementary to the target DNAtemplate; andthe forward and reverse amplification primers optionally have a combinedlength greater than the length of the target DNA template; andb) incubating the mixture partitions under thermal cycling conditionssuitable for amplification of the target DNA template by a polymerasechain reaction, wherein the thermal cycling conditions comprise a firstset of temperature cycles and a second set of temperature cycles,wherein the second set of temperature cycles comprises an annealingtemperature that is at least 5° C. higher than an annealing temperatureof the first set of temperature cycles; andc) detecting the presence or absence of amplified target DNA template inthe mixture partitions and determining the fraction of partitions wherethe target DNA template is present; thereby quantifying the amount oftarget DNA template in the sample.

In some embodiments, the 5′ tail region of the forward or the reverseamplification primers or both have at least 15%, 20%, 25%, 30%, 35%,40%, 45%, or 50% GC content. In some embodiments, the 5′ tail region ofthe forward or the reverse amplification primers or both have between15% and 80%, 15% and 75%, 15% and 70%, 15% and 65%, 15% and 60%, 15% and50%, 15% and 45%, 15% and 40%, 20% and 80%, 20% and 75%, 20% and 70%,20% and 65%, 20% and 60%, 20% and 50%, 20% and 45%, 20% and 40%, 30% and80%, 30% and 75%, 30% and 70%, 30% and 65%, 30% and 60%, 30% and 50%, or30% and 45% GC content.

In some embodiments, the target DNA template in the partitions is at anaverage concentration of 0.001-10 copies per partition.

In some embodiments, the first cycling condition comprises 1-15, 2-15,5-15, 10-15, 1-20, 2-20, 5-20, 10-20, 15-20, 1-10, 2-10, or 5-10 cycles.

In some embodiments, the thermal cycling conditions further comprise athird set of temperature cycles comprising an annealing temperature thatis at least 5° C. higher (e.g., at least 10°, 15°, or 20°, e.g., 5-25°,5-20° C.) than the annealing temperature of the second set oftemperature cycles.

In some embodiments, the first cycling condition comprises 1-20 cycles(e.g., 1-15, 2-15, 5-15, 10-15, 2-20, 5-20, 10-20, 15-20, 1-10, 2-10, or5-10 cycles) of:

i) a denaturation step;ii) a combined primer annealing and extension step; andiii) optionally, a second annealing and extension step at a highertemperature than in the first primer annealing and extension step.

In some embodiments, the second cycling condition comprises 10-50 cycles(e.g., 10, 20, 30, 40, or 50 cycles). In some embodiments, the secondcycling condition comprises 10-50 cycles (e.g., 10, 20, 30, 40, or 50cycles) of: i) a combined primer annealing and extension step; and ii) adenaturation step.

In some embodiments, the first cycling condition comprises an annealingtemperature of less than 55° C. or 50° C. (e.g., from 50° C. to 40° C.,or from 55° C. to 40° C.).

In some embodiments, the second cycling condition comprises an annealingtemperature of at least 60° C., 55° C. or 50° C. (e.g., from 50° C. to60° C., 50° C. to 65° C., 50° C. to 68° C., 55° C. to 60° C., 55° C. to65° C., or 55° C. to 68° C.).

In some embodiments, the amplification primers hybridize to oppositestrands of the target DNA template and flank a region of the target DNAtemplate having an annealing temperature of less than 55° C. or 50° C.

In some embodiments, the amplification primers hybridize to oppositestrands of the target DNA template and flank a region of the target DNAtemplate between 1-30 (e.g., 1-10, 8-12, 8-20, 10-25) nucleotides inlength.

In some embodiments, the amplification primers hybridize to oppositestrands of the target DNA template such that 3′ ends of the primershybridize to adjacent nucleotide positions in the target DNA template,i.e. zero intervening nucleotides.

In some embodiments, the amplification primers hybridize to oppositestrands of the target DNA template and the 3′ ends of the amplificationprimers overlap when hybridized to the target DNA template. In someembodiments, the length of the overlap is 1, 2, or 3 nucleotides.

In some embodiments, the 3′ hybridization regions of the amplificationprimers are at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,17, 18, 19, 20, or more nucleotides in length.

In some embodiments, the 3′ hybridization regions of the amplificationprimers are less than 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21,22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides in length.

In some embodiments, the target DNA template is less than 100, 95, 90,85, 80, 75, 70, 65, 60, 55, 50, 45, 40, or 35 nucleotides in length. Insome embodiments, the target DNA template is between 100 and 15, 100 and25, 100 and 35, 90 and 15, 90 and 25, or 90 and 35, nucleotides inlength. In some embodiments, the target DNA template comprises a regioncomplementary to an RNA (e.g., microRNA), wherein the regioncomplementary to the RNA or microRNA is less than 50, 35, 30, 25, 22,20, 18, or 15 nucleotides in length. In some embodiments, the target DNAtemplate comprises a region complementary to an RNA (e.g., microRNA),wherein the region complementary to the RNA or microRNA is between 50and 15, 50 and 18, 50 and 20, 50 and 22, 50 and 25, 50 and 30, 50 and35, 35 and 15, 35 and 18, 35 and 20, 35 and 22, 35 and 25, or 35 and 30nucleotides in length.

In some embodiments, the 5′ tail region is at least 1, 2, 3, 4, 5, 6, 7,8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleotides inlength. In some embodiments, the 5′ tail region is between 1 and 20, 2and 20, 3 and 20, 4 and 20, 5 and 20, 1 and 15, 2 and 15, 3 and 15, 4and 15, 5 and 15, 1 and 10, 2 and 10, 3 and 10, 4 and 10, or 5 and 10nucleotides in length.

In some embodiments, incubating comprises conditions such that the 3′hybridization region of the forward or the reverse amplification primerdoes not hybridize to and prime template directed extension from asecond DNA template having a polymorphism in the region to which theamplification primer is hybridized. In some embodiments, the 3′hybridization region of the forward or the reverse amplification primercomprises a discriminatory nucleotide, wherein the discriminatorynucleotide is complementary to the target DNA template but is notcomplementary to the second DNA template having the polymorphism, andwherein the discriminatory nucleotide is at the ultimate position fromthe 3′ end of the primer or is 1, 2, 3, 4, or 5 nucleotides from the 3′end. In some embodiments, the 3′ hybridization region of the forward orthe reverse amplification primer comprises a discriminatory nucleotide,wherein the discriminatory nucleotide is complementary to the target DNAtemplate but is not complementary to the second DNA template having thepolymorphism, and wherein the discriminatory nucleotide is at a positionat least 1, 2, 3, 4, 5, 6 or more nucleotides from the 3′ end of theprimer and wherein the 3′ hybridization region of the forward or thereverse amplification primer further comprises a nucleotide that is notcomplementary to the target DNA template or the second DNA template.

In some embodiments, the 3′ hybridization region of the forward orreverse amplification primer comprises a homopolymeric region of atleast 1, 2, 3, 4, or 5 nucleotides that is complementary to ahomopolymeric region of the target DNA template. In some embodiments,the homopolymeric region of the primer is between 3 and 25 contiguousnucleotides in length. In some embodiments, the homopolymeric region is3′ of a 5′ tail region that is not complementary to the target DNAtemplate. In some embodiments, the homopolymeric region of the primer isa polythymine region. In some embodiments, the homopolymeric region ofthe primer is a polyadenine region. In some embodiments, the methodcomprises adding a homopolymeric region to the target DNA template bycontacting the target DNA template with a terminal transferase enzyme.In some embodiments, the homopolymeric region of the target DNA templateis a polythymine region.

In some embodiments, the forming of the plurality of mixture partitionscomprising the target DNA template comprises reverse transcribing atarget RNA template to form the target DNA template. In someembodiments, reverse transcribing the target RNA template compriseshybridizing a reverse transcription primer to the target RNA template,wherein the target RNA template is polyadenylated at the 3′ end, and thereverse transcription primer comprises from 3′ to 5′:

i) a 3′ hybridization region that hybrids to the target RNA templatenucleic acid; and one of:ii) a homopolymeric region that is complementary to a region of thepolyadenylated 3′ end of the target RNA template; oriii) a region that is homopolymeric except has one or two nucleotidesthat are different from remaining nucleotides in the region, wherein theregion, except for said one or two nucleotides (which are bounded oneither side by homopolymeric sequence), is complementary to a region ofthe polyadenylated 3′ end of the target RNA template.

In some embodiments, the homopolymeric region is a polythymine region.In some embodiments, the homopolymeric region is a polyadenine region.In some embodiments, the homopolymeric region is a polycytosine region.In some embodiments, the homopolymeric region is a polyguanine region.In some embodiments, the homopolymeric region is from 2 to 15nucleotides in length. In some embodiments, reverse transcribing thetarget RNA template comprises hybridizing a reverse transcription primerto the target RNA template and generating a cDNA, wherein the target RNAtemplate is polyadenylated at the 3′ end and the cDNA has acomplementary polyT sequence at the 5′ end of the cDNA, and the methodfurther comprises adding a homopolymeric region to the 3′ end of thecDNA, thereby generating a cDNA having homopolymeric sequences at 5′ and3′ ends.

Also separately provided is a method of generating and amplifying cDNA.This method can optionally also be combined with the methods describedabove or elsewhere herein. In some embodiments, the method comprises

a. reverse transcribing a target RNA comprising a non-polyA region and apolyA tail by:i. hybridizing a reverse transcription (RT) primer to the target RNA,wherein the RT primer comprises from 5′ to 3′:

X-(T)_(m)-Y-(T)_(n)-Z   (SEQ ID NO:1)

-   -   wherein X is an optional (i.e., may be absent) 5′ tail        nucleotide sequence of 1-10 (e.g., 1-5 or 2-5) nucleotides that        is not complementary to the target RNA;        T is thymine;        Y is a single nucleotide that is C, G, or A;        Z is an optional sequence of 1-10 (e.g., 1-5, 2-5, or 2-10)        nucleotides that is complementary to a portion of the non-polyA        region adjacent to the polyA tail;        m is 1-20 (e.g., 1-10 or 2-10);        n is 1-20 (e.g., 1-10 or 2-10);

ii. reverse transcribing the target RNA by extending the RT primer witha RNA-dependent polymerase to generate a cDNA incorporating the RTprimer; and

b. amplifying the cDNA by:

i. hybridizing an amplification primer comprising a sequencecomplementary to the -(T)_(m)-Y-(T)_(n)-Z; and

ii. extending the amplification primer with a DNA-dependent polymerase.

In some embodiments, m=1-4 or 2-4, n=1-4 or 2-4, or both m and n areindependently 1-4 or 2-4. In some embodiments, the RT primer comprises Z(1-10 (e.g., 1-5, 2-5, or 2-10) nucleotides that are complementary to aportion of the non-polyA region adjacent to the polyA tail) and theamplification primer comprises one or more 3′ nucleotides adjacent tosequence complementary to the -(T)_(m)-Y-(T)_(n) that are complementaryto Z.

In some embodiments, the amplifying comprises a polymerase chainreaction (e.g., including but not limited to a digital PCR reaction).

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A-1B illustrates a schematic example of primers P1 and P2 used toamplify a target DNA. P1 has a 5′ tail region (A) that is notcomplementary to the target DNA and a 3′ region (B) that iscomplementary to the target DNA. P2 has a 5′ tail region notcomplementary to the target DNA and a 3′ region complementary to theopposite strand of the target DNA (SEQ ID NOs:13-14). The target DNA isshown as a block. It will be appreciated that the target DNA can startas a single-stranded or double-stranded nucleic acid but that uponamplification, the amplicon will be double stranded. As shown in FIGS.1A and 1B, a first set of cycles of amplification are performed as alower annealing and extension temperature followed by a second set ofcycles at a higher annealing and extension temperature.

FIG. 2 illustrates a schematic example in which the amplifications areperformed on a sample having a target DNA and a non-target DNA thatdiffers from the target DNA by one nucleotide (shown as the target DNAhaving a “C” and the non-target DNA having a “T”). As in FIG. 1, thetarget DNA is shown as a box and could be single- or double stranded,with the resulting amplicon being double-stranded. The “X” through thenon-target DNA indicates that sequence is not amplified because the 3′region of primer P1 is complementary to the target DNA and theconditions used for amplification do not allow for significantamplification of the non-target DNA.

FIG. 3A-3C schematically depict an embodiment of the method in which apolyA sequence is present on, or added to, an RNA molecule, which issubsequently reverse transcribed to generate the target DNA. FIG. 3Ashows the reverse transcription step with primer P1 that includes a 3′region (A) that is complementary to the RNA (SEQ ID NO:16), ahomopolymeric polyT region that is complementary to the polyA sequenceof the RNA, and may or may not include a non-homopolymeric nucleotidesomewhere within the homopolymeric region of the primer (B). Reversetranscription results in the bottom molecule in FIG. 3A. In manyembodiments, the RT primer does not include the nucleotide beingdiscriminated. FIG. 3B illustrates the first set of amplification cyclesusing the cDNA as a target DNA, with primers P1 and P2 being used toamplify the target with the complementary region (A) of the primersannealing during this set of amplification. FIG. 3C illustrates thesecond set of amplification cycles with higher temperature annealing andextension temperatures with both region A and B of the primers willanneal and amplify.

FIG. 4A-4C schematically illustrate additional aspects in which ahomopolymeric sequence is added to the initial cDNA 3′ end (e.g., byterminal transferase). In the embodiment shown, polyT is added to thecDNA in FIG. 4A (SEQ ID NO:18 (poly-T)), though other nucleotidehomopolymers (e.g., polyA, polyU, polyG, polyC) can be added. FIG. 4Billustrates the first set of amplification cycles using the cDNA as atarget DNA, with primers P1 and P2 being used to amplify the target,with P2 including a homopolymeric segment that is complementary to thehomopolymer on the cDNA sequence (SEQ ID NO:18 (poly-T) and SEQ ID NO:19(poly-A)). FIG. 4C illustrates the second set of amplification cycleswith higher temperature annealing and extension temperatures.

FIG. 5A-5C schematically illustrate additional aspects in which ahomopolymeric sequence (SEQ ID NO:18 (poly-T)) is added to the initialRNA 3′ end by using a non-enzymatic reaction (e.g., by tailed reversetranscription primer). In the embodiment shown, polyT is added to thecDNA using a tailed reverse transcription primer in FIG. 5A, thoughother nucleotides and other homopolymers (e.g., polyA, polyU, polyG,polyC) can be added. FIG. 5B illustrates the first set of amplificationcycles using the cDNA as a target DNA, with primers P1 and P2 being usedto amplify the target, with P1 including a homopolymeric segment that iscomplementary to the homopolymer on the cDNA sequence. FIG. 5Cillustrates the second set of amplification cycles with highertemperature annealing and extension temperatures (SEQ ID NO:18 (poly-T)and SEQ ID NO:19 (poly-A)).

FIG. 6 illustrates hypothetical use of polyadenylase to a target RNA.SEQ ID NOs are as follows: PolyA reaction: Target RNA (top)=SEQ IDNO:20, Target RNA (bottom)=SEQ ID NO:21; RT reaction: Target RNA=SEQ IDNO:21, RT primer=SEQ ID NO:22, cDNA product=SEQ ID NO:23; PCR reaction:primers (SEQ ID NOS:24-25), PCR products (SEQ ID NO:26-27).

FIG. 7 illustrates a second hypothetical example in which a tailedprimer is used. SEQ ID NOs are as follows: RT reaction: Target RNA=SEQID NO:20, RT primer=SEQ ID NO:32, cDNA product=SEQ ID NO:23; PCRreaction: primers (SEQ ID NOS:24-25), PCR products (SEQ ID NO:26-27).

FIG. 8 illustrates a hypothetical example involving introduction of ahomopolymeric sequence using terminal transferase. SEQ ID NOs are asfollows: RT reaction: Target RNA=SEQ ID NO:21, RT primer=SEQ ID NO:28,cDNA product=SEQ ID NO:29; Terminal Transferase Reaction: topsequence=SEQ ID NO:30, Terminal Transferase=SEQ ID NO:31, PCR reaction:primers (SEQ ID NOS:24 and 33), PCR products (SEQ ID NO:34-35).

DEFINITIONS

The term “polymerase” refers to an enzyme that performstemplate-directed synthesis of polynucleotides. The term encompassesboth a full length polypeptide and a domain that has polymeraseactivity. DNA polymerases are well-known to those skilled in the art,and include but are not limited to DNA polymerases isolated or derivedfrom Pyrococcus furiosus, Thermococcus litoralis, and Thermotogamaritime, or modified versions thereof. They include both DNA-dependentpolymerases and RNA-dependent polymerases such as reverse transcriptase.At least five families of DNA-dependent DNA polymerases are known,although most fall into families A, B and C. There is little or nosequence similarity among the various families. Most family Apolymerases are single chain proteins that can contain multipleenzymatic functions including polymerase, 3′ to 5′ exonuclease activityand 5′ to 3′ exonuclease activity. Family B polymerases typically have asingle catalytic domain with polymerase and 3′ to 5′ exonucleaseactivity, as well as accessory factors. Family C polymerases aretypically multi-subunit proteins with polymerizing and 3′ to 5′exonuclease activity. In E. coli, three types of DNA polymerases havebeen found, DNA polymerases I (family A), II (family B), and III (familyC). In eukaryotic cells, three different family B polymerases, DNApolymerases α, δ, and ϵ, are implicated in nuclear replication, and afamily A polymerase, polymerase γ, is used for mitochondrial DNAreplication. Other types of DNA polymerases include phage polymerases.Similarly, RNA polymerases typically include eukaryotic RNA polymerasesI, II, and III, and bacterial RNA polymerases as well as phage and viralpolymerases. RNA polymerases can be DNA-dependent and RNA-dependent. Thepolymerases described herein can be heterologous to the target nucleicacid(s) in a reaction mixture, mixture partition, or set of mixturepartitions. As used herein, the term “heterologous” refers to twocomponents (e.g., target nucleic acid and polymerase) that are not foundtogether in nature, e.g., because they are not found together in thesame wild-type organism.

“Thermally stable polymerase,” as used herein, refers to any enzyme thatcatalyzes polynucleotide synthesis by addition of nucleotide units to anucleotide chain using DNA or RNA as a template and has an optimalactivity at a temperature above 45° C.

The term “nucleic acid amplification” or “amplification reaction” refersto any in vitro means for multiplying the copies of a target sequence ofnucleic acid. Such methods include but are not limited to polymerasechain reaction (PCR), DNA ligase chain reaction (see U.S. Pat. Nos.4,683,195 and 4,683,202; PCR Protocols: A Guide to Methods andApplications (Innis et al., eds, 1990)), (LCR), QBeta RNA replicase, andRNA transcription-based (such as TAS and 3SR) amplification reactions aswell as others known to those of skill in the art.

“Amplifying” refers to a step of submitting a solution to conditionssufficient to allow for amplification of a polynucleotide. Components ofan amplification reaction include, e.g., primers, a polynucleotidetemplate, polymerase, nucleotides, and the like. The term amplifyingtypically refers to an “exponential” increase in target nucleic acid.However, amplifying as used herein can also refer to linear increases inthe numbers of a select target sequence of nucleic acid, such as isobtained with cycle sequencing.

“Polymerase chain reaction” or “PCR” refers to a method whereby aspecific segment or subsequence of a target double-stranded DNA, isamplified in a geometric progression. PCR is well known to those ofskill in the art; see, e.g., U.S. Pat. Nos. 4,683,195 and 4,683,202; andPCR Protocols: A Guide to Methods and Applications, Innis et al., eds,1990. Exemplary PCR reaction conditions typically comprise either two orthree step cycles. Two step cycles have a denaturation step followed bya hybridization/elongation step. Three step cycles comprise adenaturation step followed by a hybridization step followed by aseparate elongation step. PCR can be performed as end-point PCR (i.e.,only monitored at an end point) or as quantitative PCR (monitored in“real time”).

An “olignucleotide primer” or “primer” refers to an oligonucleotidesequence that anneals to a sequence on a target nucleic acid and servesas a point of initiation of nucleic acid synthesis.

The terms “nucleic acid” and “polynucleotide” are used interchangeablyherein to refer to deoxyribonucleotides or ribonucleotides and polymersthereof in either single- or double-stranded form. The term encompassesnucleic acids containing known nucleotide analogs or modified backboneresidues or linkages, which are synthetic, naturally occurring, andnon-naturally occurring, which have similar binding properties as thereference nucleic acid, and which are metabolized in a manner similar tothe reference nucleotides. Examples of such analogs include, withoutlimitation, phosphorothioates, phosphoramidates, methyl phosphonates,chiral-methyl phosphonates, 2-O-methyl ribonucleotides, and peptidenucleic acids (PNAs).

The terms “polypeptide,” “peptide,” and “protein” are usedinterchangeably herein to refer to a polymer of amino acid residues. Theterms apply to amino acid polymers in which one or more amino acidresidue is an artificial chemical mimetic of a corresponding naturallyoccurring amino acid, as well as to naturally occurring amino acidpolymers and non-naturally occurring amino acid polymers.

The term “amino acid” refers to naturally occurring and synthetic aminoacids, as well as amino acid analogs and amino acid mimetics thatfunction in a manner similar to the naturally occurring amino acids.Naturally occurring amino acids are those encoded by the genetic code,as well as those amino acids that are later modified, e.g.,hydroxyproline, γ-carboxyglutamate, and O-phosphoserine. Amino acidanalogs refers to compounds that have the same basic chemical structureas a naturally occurring amino acid, i.e., an a carbon that is bound toa hydrogen, a carboxyl group, an amino group, and an R group, e.g.,homoserine, norleucine, methionine sulfoxide, methionine methylsulfonium. Such analogs have modified R groups (e.g., norleucine) ormodified peptide backbones, but retain the same basic chemical structureas a naturally occurring amino acid. Amino acid mimetics refers tochemical compounds that have a structure that is different from thegeneral chemical structure of an amino acid, but that functions in amanner similar to a naturally occurring amino acid.

Amino acids may be referred to herein by either their commonly knownthree letter symbols or by the one-letter symbols recommended by theIUPAC-IUB Biochemical Nomenclature Commission. Nucleotides, likewise,may be referred to by their commonly accepted single-letter codes.

The term “partitioning” or “partitioned” refers to separating a sampleinto a plurality of portions, or “partitions.” Partitions can be solidor fluid. In some embodiments, a partition is a solid partition, e.g., amicrochannel. In some embodiments, a partition is a fluid partition,e.g., a droplet. In some embodiments, a fluid partition (e.g., adroplet) is a mixture of immiscible fluids (e.g., water and oil). Insome embodiments, a fluid partition (e.g., a droplet) is an aqueousdroplet that is surrounded by an immiscible carrier fluid (e.g., oil).

DETAILED DESCRIPTION OF THE INVENTION I. Introduction

Methods and reaction mixtures have been discovered for the amplificationof RNA targets, and particularly, short RNA targets such as, but notlimited to, micro RNAs. The RNA target is initially reverse transcribedinto a target DNA. In other embodiments, the methods and reactionmixtures can be used to amplify a target DNA (e.g., a DNA other thancDNA). The methods involve using a first and a second set ofamplification cycles (e.g., PCR cycles) to amplify the target DNA, inwhich the second set of cycles comprise an annealing temperature at ahigher temperature than the annealing temperature of the first set ofconditions. In addition, forward and reverse amplification primers areprovided wherein the primers both have a 3′ region complementary to thetarget DNA and at least one (or both) of the primers have a 5′ tail thatis not complementary to the target. The first set of cycles is performedat an annealing temperature to allow for amplification based onhybridization of the 3′ regions of the primers to the target DNA.Following a number of cycles (e.g., 5-10 or 5-15) in the first set, anamplicon is established that incorporates the 5′ tail(s) of the primers,thereby forming a longer amplicon to which the tailed primer(s)hybridize with a higher Tm, allowing for the second set of cycles tohave a higher annealing and extension temperature at which thepolymerase functions better (closer or at optimal).

Also provided is a method of reverse transcribing a polyadenylated(polyA) RNA into a cDNA and amplifying the cDNA using a reversetranscription primer that comprises a polyT sequence containing one ortwo intervening nucleotides that are nucleotides other than T, therebygenerating a cDNA having a modified polyT sequence comprising the one ortwo intervening nucleotides. The cDNA can then be amplified using aprimer complementary to the modified polyT sequence. The inventors havefound this method results in higher specificity and sensitivity thanusing an otherwise identical primer but lacking the interveningnucleotides.

Exemplary target RNAs that can be detected and amplified using themethods described herein include, but are not limited to, miRNA, snRNA,snoRNA, piRNA, or lncRNA. MicroRNAs (miRNAs), typically 18 to 25 nt inlength, are non-protein-coding RNAs that can inhibit the translation oftarget mRNAs (see, e.g., Croce and Calin, Cell 122(1): 6-7 (2005)).Other small RNAs include small nucleoplasmic RNAs (snRNAs) and smallnucleolar RNAs (snoRNAs). These small RNA molecules can function, forexample, in mRNA splicing (U1, U2, and U4 to U6 snRNAs), mRNA and rRNAprocessing (U7 snRNA; U3 and U8 snoRNAs), and site selection for RNAmodification by methylation of the 2′ hydroxyl group (box C/D snoRNAs)or by pseudouridine formation (box H/ACA snoRNAs). Piwi-interacting RNAs(piRNAs) were identified through association with Piwi proteins inmammalian. piRNAs can range from 26-30 nucleotides in length. Longnoncoding RNA (lncRNA) have also been described.

Additional aspects of the inventions are provided herein.

II. Reaction Components

Reaction components can include a sample comprising target DNA and aforward primer and reverse primer as described herein.

The methods described herein can be performed as one or more polymerasechain reaction (PCR) amplification. The methods are particularly usefulin partitioned PCR methods such as digital PCR. Thus in someembodiments, a plurality of reaction mixtures are prepared each having alow average copy number of target DNA. For example, in some embodiments,the average target DNA copy number is 0.001-10 copies per partition(e.g., 0.01, 0.05, 0.1, 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 copies perpartition). The number of partitions can vary as understood in the artbut can range, for example, from 1000-10¹⁰ partitions or more.

The reaction mixture will comprise at least two oligonucleotide primers,referred here only to differentiate them as “forward” and “reverse”primers. Each primer will have a 3′ region that hybridizes to the targetDNA. The 3′ region of the primers can be, for example, 3-20 nucleotideslong, (e.g., 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more, e.g.,5-15, 8-12, 5-20, etc.). In some embodiments, the 3′ regions are fullycomplementary to the corresponding target DNA sequence. In otherembodiments, the 3′ region can have at most one or two mismatches withthe target DNA sequence. As discussed above and elsewhere herein, themethods involve employing at least two different sets of cycles ofamplification with the second set of cycles having a warmer annealingtemperature than the first set. The 3′ regions of the primers can beselected to have a melting temperature (Tm) close to, for example, at orbelow the second annealing temperature. For example, in someembodiments, the Tm of one or both 3′ regions is approximately the sameas or within 5, 10, 15, 20, 25, or 30 (e.g., 3-20, 3-25) degrees C.lower than the annealing temperature of the second set of cycles.

FIGS. 1A-B show an example of some of the different components of themixture. Primers P1 and P2 are shown. Primer regions B and C are the 3′regions of the primers P1 and P2, respectively. It will be appreciatedthat primer P1 hybridizes to one strand of the target DNA and primer P2hybridizes to the opposing strand of the target DNA. In cases where theinitial target DNA is single-stranded, the amplicon after the firstcycle will include a second strand.

The forward primer, reverse primer, or both, can also have a 5′ tailregion that does not hybridize to the target DNA. This 5′ tail regioncan function to lengthen the amplicon in rounds of amplification afterthe initial amplification rounds. For example, initial rounds ofamplification will result in an amplicon comprising the target DNAsequence as well as the 5′ tail region. The top section of FIG. 1illustrates one embodiment in which the 3′ regions of the primershybridize to the target DNA while the 5′ regions do not. Sequence of the5′ tail region can be selected so that the Tm of the primer as a whole(3′ region and 5′ region) has a Tm at or below the extension temperatureof the second set of amplification cycles. In some embodiments, the Tmof the primer as a whole is approximately the same as or within 5, 10,15, 20, 25, or 30 degrees C. lower than the annealing temperature of thesecond set of cycles. The sequences of the forward and reverse primerswill generally be selected to avoid primer dimer formation or forself-hybridization of primers. In some embodiments, the 5′ regions areselected to have a GC content of greater than 30, 40, 50, 60, 70, 80,85, 90, or 95%, e.g., 100%. The length of the 5′ tail region can vary asdesired. For example, in some embodiments, the 5′ tail region ii 1-20nucleotides long, e.g., 1, 2, 3, 4, 5, 7, 10 nucleotides, etc.). In someembodiments, the 5′ tail portion (not counting the remaining of theprimer) has a Tm at or lower than the first annealing temperature.

In some embodiments, a target DNA will be highly similar to a non-targetDNA sequence. For example, where the target DNA is a cDNA from amicroRNA to be detected but a non-target microRNA is highly similar, acontaminating cDNA from the non-target microRNA can interfere withdetection. To reduce false positives, in some embodiments, the 3′ regionof one or both primers is selected so that the 3′ region is not fullycomplementary (e.g. at least one or two nucleotides are notcomplementary) to the non-target DNA. In some embodiments, thenon-complementary nucleotide(s) (relative to the non-target DNA), alsoreferred to herein as “discriminatory nucleotides,” is located at the 3′end of the primer or is 1, 2, 3, 4, or 5 nucleotides from the 3′ end.Because a DNA target can be very short, in some embodiments, thenon-complementary nucleotide can be positioned farther than 5nucleotides from the 3′ end of the primer. This aspect is depicted inFIG. 2 where two DNA sequences, one containing the target C and theother DNA sequence comprising a T at the same position. Primer P1comprises a 3′ region completely complementary to the target DNA (i.e.,has a G to complement the target C), but has a non-complementarynucleotide with regard to the non-target DNA. Annealing and extensionconditions can then be selected such that the primer hybridizes to thetarget but does not hybridize to the non-target. “Does not hybridize” inthis context means that the target DNA is amplified at least 10 times,and in some embodiments, at least 100 or 1000 times the amount thenon-target DNA is amplified.

In some cases, it can be helpful to further select a nucleotide inanother location in the 3′ region of the primer to mis-match with boththe target and non-target DNA. This will reduce the Tm of the primer forthe target but the Tm for the non-target DNA will be even lower becausethe 3′ region will be mismatched at two locations. By setting theannealing temperature below the Tm for the target and above the Tm forthe non-target DNA, the target can be amplified with little or nonon-target DNA amplified.

Positioning of the area of hybridization of the forward and reverseprimers on the target DNA will be a function, in part, of the 3′ regionof the primers. Thus, the 3′ region of the forward primer will have abinding site on the target DNA and the 3′ region of the reverse primerwill have a binding site on the target DNA (on opposing strands of adouble stranded target DNA). In some embodiments, two binding sites willhave one or more nucleotides (e.g., 1-25, 1-10, 8-12 nucleotides)between the binding sites in the target DNA. In other embodiments, thebinding sites will be adjacent (no nucleotides between the two bindingsites is shown in FIG. 1A-B where the gap is “0”). In yet anotherembodiment, the binding sites of the two primers will be overlapping.For example, the binding sites can overlap by 1, 2, 3, 4 or more (e.g.,1-3) nucleotides. This aspect is depicted in FIG. 1 where the overlap isindicated a being “0-3,” meaning that in some embodiments, the bindingsites overlap by 3 nucleotides, in other embodiments the binding sitesoverlap by 2 nucleotide, in other embodiments the binding sites overlapby 1 nucleotide or are adjacent (“0” gap).

In some embodiments, the target DNA will further comprise ahomopolymeric sequence on the 5′ end, 3′ end, or both. Homopolymericsequences are sequences of at least 2, 3, 4, 5, 6, 7, 8, 9, 10, or more(e.g., 3-10, 3-20, 3-50) adjacent identical nucleotides. Exemplaryhomopolymeric sequences include polyA, polyT, polyG, polyC, or polyUsequences. In embodiments in which the target DNA has one or morehomopolymeric sequence the corresponding primer for amplification of thetarget can have a complementary homopolymeric sequence. Exemplaryaspects are illustrated, for example in FIGS. 3A-C, 4A-C and 5A-C. Thehomopolymeric sequence in the primers need not be the same length as thehomopolymeric sequence in the target DNA. For example, the primerhomopolymeric sequence can be shorter than the target homopolymericsequence. In some embodiments, one or both primers will havehomopolymeric sequences complementary to homopolymeric sequences in thetarget DNA and one or more of the primers will further comprise a 5′tail region that is not complementary to the target DNA.

Target DNA can be generated from any biological sample. An advantage ofthe present methods is the ability to amplify short sequences and thusin some embodiments, the target DNA is less than 90, 80, 70, 60, 50, 40,35 or 30 nucleotides, for example, between 10-30, 20-30, 20-40, 20-50,or 10-90 nucleotides in length. In many embodiments, the DNA is a cDNAfrom an RNA present in a sample. The sample can be for example, anymixture containing a short RNA. In many embodiments, the sample isderived from a biological fluid, cell or tissue. The sample can be crudeor purified. In some cases, the sample is a preparation of RNA from acell or cells. In some embodiments, the cells are animal cells,including but not limited to, human, or non-human, mammalian cells.Non-human mammalian cells include but are not limited to, primate cells,mouse cells, rat cells, porcine cells, and bovine cells. In someembodiments, the cells are plant or fungal (including but not limited toyeast) cells. Cells can be, for example, cultured primary cells,immortalized culture cells or can be from a biopsy or tissue sample,optionally cultured and stimulated to divide before assayed. Culturedcells can be in suspension or adherent prior to and/or during thepermeabilization and/or DNA modification steps. Cells can be from animaltissues, biopsies, etc. For example, the cells can be from a tumorbiopsy.

In some embodiments, samples include RNA or DNA targets that only haveshort amplifiable regions (e.g., wherein a target region has less than50, 40, 30, 25, or 20 contiguous amplifiable nucleotides), degraded, orare otherwise difficult to amplify due to nucleic acid degradation. Forexample, formalin-fixed samples can have only short sequences of nucleicacid due to fixation. In other embodiments, ancient nucleic acid samplesor samples that have been exposed to chemical or temperature conditionsthat degrade nucleic acids can be amplified by the methods describedherein in view of the method's ability to amplify shorter sequences thantypically can be amplified in PCR.

In other embodiments, the same basic components of the reaction mixtureas described above can be used to amplify any cDNA or other DNAmolecule, whether longer or shorter. These aspects are of particularinterest when distinguishing a target DNA from a different non-targetDNA that differs by a single nucleotide. In these aspects, the 3′ regionof one primer (e.g., one which also has a 5′ tail sequence as describedabove) is relatively short (e.g., 3-18, 5-19, 8-22, 3-8, 5-10nucleotides) with one of the nucleotides in the 3′ region beingcomplementary to the nucleotide in the target DNA that differs in thenon-target DNA. By selecting a shorter 3′ region, the difference in Tmfor the target DNA compared to the non-target DNA will be accentuated,thus allowing for a relatively short complementary region to distinguishbetween target and non-target DNA. Additionally this method is not proneto the DNA polymerase's misreading or error rates during extension forthe discrimination since this method uses a non-hybridized 3′ nucleotideto prevent the extension step from beginning on non-target templates.After a first set of cycles at this lower Tm for the target DNA, theresulting amplicon will incorporate the 5′ tail sequence(s) and thus asecond set of amplification cycles at a higher Tm can be used asdescribed elsewhere herein. Because this aspect can be used for longertarget sequences, the primers can be designed to accommodate a labeledprobe (e.g., a Taqman or molecular beacon probe) if desired. In otherembodiments, an intercalating dye as described elsewhere herein can beused to detect the amplification product.

III. Methods

The methods described herein provide for amplification with at least twodifferent sets of amplification cycles where the second set ofamplification cycles has an annealing temperature higher than the firstset of cycles. In some embodiments, the second set of amplificationcycles has an annealing temperature at least 1, 2, 3, 4, 5, 6, 7, 8, 9,or 10 (e.g., 5-15) degrees C. higher than the first set of amplificationcycles. In some embodiments, the first set of cycles employ an annealingtemperature of less than, e.g., 55, 50, 48, or 45° C. In someembodiments, the second set of cycles employ an annealing temperature ofmore than, e.g., 50, 55, or 60° C. (e.g., 50-75° C.).

In some embodiments, the number of cycles in the first set of cycleswill be smaller than the number of cycles in the second set. In part,this can be because the first set of cycles generates the initialamplicon comprising the incorporated primers, after which the second setof cycles can then continue to amplify the enlarged amplicon withgreater efficiency. In some embodiments, the first set of cycles willhave between 3-20 cycles, e.g., 5-15 cycles. In some embodiments, thesecond set of cycles will have at least 15 cycles, e.g., between 15-45,15-40, 20-40, 25-35 cycles.

An amplification “cycle” refers to a series of temperature changes thatsupport denaturation of double stranded DNA, annealing of primers to thetarget DNA, and extension of the primers by a DNA polymerase. Typically,a three-step or two-step cycle is used. A three-step cycle comprises aseparate denaturation step (e.g., 90-98 degrees C.), a separateannealing step (e.g., 50-65 degrees C.), and a separate extension step(e.g., 65-75 degrees C.). In a two-step cycle, a denaturation step asdescribed above is followed by a combined annealing/extension step(e.g., 50-65 degrees C.). While this document refers to the second setof cycles having a higher annealing temperature than the first set, itshould be understood that in a two-step cycle, the extension step of thesecond set of cycles will also necessarily be higher than in the firstset of cycles. In a three-step cycle, the extension step can be but doesnot necessarily have to be higher in the second set of cycles comparedto the extension step in the first set of cycles.

As noted above, one use of the described methods is for detection andquantifying small RNAs including but not limited to miRNA, snoRNA,piRNA, or lncRNA. As such, prior to the steps described above, a reversetranscription reaction can be performed to generate a cDNA. Reversetranscription can be performed as desired using a reverse transcriptaseto generate a cDNA. Any of a variety of reverse transcriptases can beused. Exemplary reverse transcriptases include but are not limited tomurine leukemia virus (MLV) reverse transcriptase, Avian MyeloblastosisVirus (AMV) reverse transcriptase, Respiratory Syncytial Virus (RSV)reverse transcriptase, Equine Infectious Anemia Virus (EIAV) reversetranscriptase, Rous-associated Virus-2 (RAV2) reverse transcriptase,SUPERSCRIPT II reverse transcriptase, SUPERSCRIPT I reversetranscriptase, THERMOSCRIPT reverse transcriptase and MMLV RNase H⁻reverse transcriptases. In additional embodiments, a DNA polymerase thatfunctions as an RNA polymerase can be used. For example, Tth and Z05,which are DNA polymerases, can function as reverse transcriptase in thepresence of manganese. The concentration of the reverse transcriptasecan vary and optimal concentrations can be determined empirically anddepend on the particular reverse transcriptase used.

In some embodiments, the RNA from the sample of interest contains apolyA tail. However, many small RNAs do not necessarily have a polyAtail. In some embodiments, a polyA tail can be added to an RNA prior togeneration of the cDNA. For example, the RNA can be incubated withpoly(A) polymerase and ATP to polyadenylate the RNA. This step can thenbe followed by reverse transcription as described herein.

Once a target DNA is available, one can add a homopolymeric 3′ sequence,for example by ligation or incubating the DNA with terminal transferaseand a single nucleotide (e.g., dTTP, dATP, dGTP, dCTP, dUTP). Theresulting DNA molecule will comprise the original target DNA sequenceand a 3′ homopolymer. In some embodiments, a homopolymeric sequence willbe added to both ends of the target DNA using one or more of the methodsdescribed herein (e.g., polyadenylation of the RNA and a polyT sequencecan be added to the DNA with terminal transferase). In embodiments wherea homopolymeric sequence has been added to the RNA, the resultingtemplate for amplification will be longer, making design of primers foramplification easier and allowing for higher temperature for annealingin amplification. Also poly adenylation or other methods of lengtheningthe cDNA can allow for the discrimination of the target by providingenough sequence to place the 3′ end of the PCR primer over thenucleotide(s) to be discriminated within 1, 2, 3, 4 or 5 nucleotides ofa primer's 3′ end.

Also provided is a method of reverse transcribing an RNA having a polyAtail. This method can be performed separate from the above describedsplit-cycle method (e.g., for any reverse transcription purpose) or canbe used in conjunction with the split-cycle method described herein. Insome embodiments, the RNA will naturally comprise a polyA tail (forexample, mRNA). In other aspects the polyA tail can be added to an RNAusing poly A polymerization. See, e.g., Cao, G. J. and Sarkar, N. Proc.Natl. Acad. Sci. USA. 89, 10380-10384 (1992).

Once a polyA-containing RNA is provided, the RNA can be reversetranscribed using a reverse transcription (RT) primer wherein the RTprimer comprises from 5′ to 3′:

X-(T)_(m)-Y-(T)_(n)-Z   (SEQ ID NO:1)

-   -   wherein X is an optional 5′ tail nucleotide sequence of 1-10        nucleotides that is not complementary to the target RNA;    -   T is Thymine;    -   Y is a single nucleotide that is C, G, or A;    -   Z is an optional sequence of 1-10 nucleotides that is        complementary to a portion of the non-polyA region adjacent to        the polyA tail;    -   m is 1-10 (e.g., 2-10, 2-5); and    -   n is 1-10 (e.g., 2-10, 2-5).

The above-described primer does not have a single homopolymeric polyTsequence complementary to the polyA sequence of the RNA and instead hasa polyT sequence divided by (having polyT on both side of) one or twonucleotides other than T. Thus, when the RT primer hybridizes to thepolyA RNA, the polyT portions of the RT primer will hybridize to thepolyA portion of the RNA, but the one or two nucleotides other than Twill not be complementary to the polyA portion and will form a “bulge”and area of non-hybridization. Because RT conditions are selected toallow for hybridization nevertheless, the primer will prime the RTreaction to generate a cDNA. Notably, the resulting cDNA will contain atits 5′ end a complementary sequence to the one or two nucleotides. Thiswill allow for much more specific amplification of the cDNA insubsequent amplification that uses a primer complementary to the-(T)_(m)-Y-(T)_(n) sequence. As an Example, if the RT primer was5′tttttCtttttacgc (i.e., m=5, Y=C, n=5; (SEQ ID NO:2)), 3′ a PCR primercould be: 5′gcgtaaaaagaaaaa 3′ (SEQ ID NO:3).

In some embodiments, the RT primer will include one or more 3′nucleotides that specifically hybridize to the 3′ end of the non-polyAportion of the target RNA, thereby making the RT primer more specific.For instance, in the above example, “acgc” is a sequence complementaryto the last four non-polyA nucleotides of a target RNA (and accordinglythe PCR primer has a complementary “gcgt” sequence).

The amplification reactions described herein can be detected as desired.In some embodiments, a label that generates signal in the presence ofdouble-stranded DNA is used. In some embodiments, intercalating agentsthat produce a signal when intercalated in double stranded DNA may beused. Exemplary agents include SYBR GREEN™, SYBR GOLD™, and EVAGREEN™.Since these agents are not template-specific, it is assumed that thesignal is generated based on template-specific amplification. This canbe confirmed, if desired, by monitoring signal as a function oftemperature because melting point of template sequences will generallybe much higher than, for example, primer-dimers, etc.

Detection and quantification of target DNAs is preferably carried out bydigital PCR. In general aspects, digital PCR is carried out bypartitioning a dilute sample into a plurality of discrete partitionssuch that most of the plurality of discrete test sites comprise onaverage a low number of initial target DNA copies. Amplificationproducts are then analyzed and quantified, resulting in a representationof the presence or absence of genomic regions of interest correspondingto the presence or absence of the target DNA. The number of target DNA(and thus target RNA) copies can then be quantified to estimate thenumber of target DNA copies in a sample. The number of partitions mayvary depending on the application and the level of statisticalconfidence to be achieved.

The separated sample comprising target DNA (or RNA if reversetranscription is to occur in the partitions) can be partitioned into aplurality of partitions. Partitions can include any of a number of typesof partitions, including solid partitions (e.g., wells or tubes) andfluid partitions (e.g., aqueous droplets within an oil phase). In someembodiments, the partitions are droplets. In some embodiments, thepartitions are microchannels. Methods and compositions for partitioninga sample are described, for example, in published patent applications WO2010/036352, US 2010/0173394, US 2011/0092373, US2014/0170736, and US2011/0092376, the entire content of each of which is incorporated byreference herein.

In some embodiments, the sample is partitioned into a plurality ofdroplets. In some embodiments, a droplet comprises an emulsioncomposition, i.e., a mixture of immiscible fluids (e.g., water and oil).In some embodiments, a droplet is an aqueous droplet that is surroundedby an immiscible carrier fluid (e.g., oil). In some embodiments, adroplet is an oil droplet that is surrounded by an immiscible carrierfluid (e.g., an aqueous solution). In some embodiments, the dropletsdescribed herein are relatively stable and have minimal coalescencebetween two or more droplets. In some embodiments, less than 0.0001%,0.0005%, 0.001%, 0.005%, 0.01%, 0.05%, 0.1%, 0.5%, 1%, 2%, 3%, 4%, 5%,6%, 7%, 8%, 9%, or 10% of droplets generated from a sample coalesce withother droplets. The emulsions can also have limited flocculation, aprocess by which the dispersed phase comes out of suspension in flakes.

In some embodiments, the droplet is formed by flowing an oil phasethrough an aqueous solution comprising the label(s) to be detected. Insome embodiments, the aqueous sample comprising the label(s) to bedetected comprises a buffered solution and reagents for detecting thelabel(s). The oil for the oil phase may be synthetic or naturallyoccurring. In some embodiments, the oil comprises carbon and/or silicon.In some embodiments, the oil comprises hydrogen and/or fluorine.Exemplary oils include, but are not limited to, silicone oil, mineraloil, fluorocarbon oil, vegetable oil, or a combination thereof.

The oil phase may comprise a fluorinated base oil which may additionallybe stabilized by combination with a fluorinated surfactant such as aperfluorinated polyether. In some embodiments, the base oil comprisesone or more of a HFE 7500, FC-40, FC-43, FC-70, or another commonfluorinated oil. In some embodiments, the oil phase comprises an anionicfluorosurfactant. In some embodiments, the anionic fluorosurfactant isAmmonium Krytox (Krytox-AS), the ammonium salt of Krytox FSH, or amorpholino derivative of Krytox FSH. Krytox-AS may be present at aconcentration of about 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%,0.9%, 1.0%, 2.0%, 3.0%, or 4.0% (w/w). In some embodiments, theconcentration of Krytox-AS is about 1.8%. In some embodiments, theconcentration of Krytox-AS is about 1.62%. Morpholino derivative ofKrytox FSH may be present at a concentration of about 0.1%, 0.2%, 0.3%,0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1.0%, 2.0%, 3.0%, or 4.0% (w/w). Insome embodiments, the concentration of morpholino derivative of KrytoxFSH is about 1.8%. In some embodiments, the concentration of morpholinoderivative of Krytox FSH is about 1.62%.

In some embodiments, the oil phase further comprises an additive fortuning the oil properties, such as vapor pressure, viscosity, or surfacetension. Non-limiting examples include perfluorooctanol and1H,1H,2H,2H-Perfluorodecanol. In some embodiments,1H,1H,2H,2H-Perfluorodecanol is added to a concentration of about 0.05%,0.06%, 0.07%, 0.08%, 0.09%, 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%,0.8%, 0.9%, 1.0%, 1.25%, 1.50%, 1.75%, 2.0%, 2.25%, 2.5%, 2.75%, or 3.0%(w/w). In some embodiments, 1H,1H,2H,2H-Perfluorodecanol is added to aconcentration of about 0.18% (w/w).

In some embodiments, the emulsion is formulated to produce highlymonodisperse droplets having a liquid-like interfacial film that can beconverted by heating into microcapsules having a solid-like interfacialfilm; such microcapsules may behave as bioreactors able to retain theircontents through an incubation period. The conversion to microcapsuleform may occur upon heating. For example, such conversion may occur at atemperature of greater than about 40°, 50°, 60°, 70°, 80°, 90°, or 95°C. During the heating process, a fluid or mineral oil overlay may beused to prevent evaporation. Excess continuous phase oil may or may notbe removed prior to heating. The biocompatible capsules may be resistantto coalescence and/or flocculation across a wide range of thermal andmechanical processing.

In some embodiments, the sample is partitioned into at least 500partitions (e.g., droplets), at least 1000 partitions, at least 2000partitions, at least 3000 partitions, at least 4000 partitions, at least5000 partitions, at least 6000 partitions, at least 7000 partitions, atleast 8000 partitions, at least 10,000 partitions, at least 15,000partitions, at least 20,000 partitions, at least 30,000 partitions, atleast 40,000 partitions, at least 50,000 partitions, at least 60,000partitions, at least 70,000 partitions, at least 80,000 partitions, atleast 90,000 partitions, at least 100,000 partitions, at least 200,000partitions, at least 300,000 partitions, at least 400,000 partitions, atleast 500,000 partitions, at least 600,000 partitions, at least 700,000partitions, at least 800,000 partitions, at least 900,000 partitions, atleast 1,000,000 partitions, at least 2,000,000 partitions, at least3,000,000 partitions, at least 4,000,000 partitions, at least 5,000,000partitions, at least 10,000,000 partitions, at least 20,000,000partitions, at least 30,000,000 partitions, at least 40,000,000partitions, at least 50,000,000 partitions, at least 60,000,000partitions, at least 70,000,000 partitions, at least 80,000,000partitions, at least 90,000,000 partitions, at least 100,000,000partitions, at least 150,000,000 partitions, or at least 200,000,000partitions.

In some embodiments, the sample is partitioned into a sufficient numberof partitions such that at least a majority of partitions have no morethan 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40, 50, 60, 70, 80, 90,100, 200, 300, 400, or 500 copies of a label. In some embodiments, amajority of the partitions have no more than 1, 2, 3, 4, 5, 6, 7, 8, 9,10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, or 500copies of the one or more labels to be detected. In some embodiments, onaverage no more than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40, 50,60, 70, 80, 90, 100, 200, 300, 400, or 500 copies of the one or morelabels are present per partition.

In some embodiments, the sample is partitioned into a sufficient numberof partitions such that, on average, at least one partition lacks a copyof the label. In some embodiments, the sample is partitioned into asufficient number of partitions such that, on average, at least 5, 10,15, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 125, 150, 175, 200,250, 300, 350, 400, 450, or 500 partitions lack a copy of the label. Insome embodiments, the sample is partitioned into a sufficient number ofpartitions such that, on average, at least 5, 10, 15, 20, 25, 30, 35,40, 50, 60, 70, 80, 90, 100, 125, 150, 175, 200, 250, 300, 350, 400,450, or 500 partitions lack a copy of the label and such that, onaverage, at least 5, 10, 15, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90,100, 125, 150, 175, 200, 250, 300, 350, 400, 450, or 500 partitions haveat least one copy of the label.

In some embodiments, the droplets that are generated are substantiallyuniform in shape and/or size. For example, in some embodiments, thedroplets are substantially uniform in average diameter. In someembodiments, the droplets that are generated have an average diameter ofabout 0.001 microns, about 0.005 microns, about 0.01 microns, about 0.05microns, about 0.1 microns, about 0.5 microns, about 1 microns, about 5microns, about 10 microns, about 20 microns, about 30 microns, about 40microns, about 50 microns, about 60 microns, about 70 microns, about 80microns, about 90 microns, about 100 microns, about 150 microns, about200 microns, about 300 microns, about 400 microns, about 500 microns,about 600 microns, about 700 microns, about 800 microns, about 900microns, or about 1000 microns. In some embodiments, the droplets thatare generated have an average diameter of less than about 1000 microns,less than about 900 microns, less than about 800 microns, less thanabout 700 microns, less than about 600 microns, less than about 500microns, less than about 400 microns, less than about 300 microns, lessthan about 200 microns, less than about 100 microns, less than about 50microns, or less than about 25 microns. In some embodiments, thedroplets that are generated are non-uniform in shape and/or size.

In some embodiments, the droplets that are generated are substantiallyuniform in volume. For example, in some embodiments, the droplets thatare generated have an average volume of about 0.001 nL, about 0.005 nL,about 0.01 nL, about 0.02 nL, about 0.03 nL, about 0.04 nL, about 0.05nL, about 0.06 nL, about 0.07 nL, about 0.08 nL, about 0.09 nL, about0.1 nL, about 0.2 nL, about 0.3 nL, about 0.4 nL, about 0.5 nL, about0.6 nL, about 0.7 nL, about 0.8 nL, about 0.9 nL, about 1 nL, about 1.5nL, about 2 nL, about 2.5 nL, about 3 nL, about 3.5 nL, about 4 nL,about 4.5 nL, about 5 nL, about 5.5 nL, about 6 nL, about 6.5 nL, about7 nL, about 7.5 nL, about 8 nL, about 8.5 nL, about 9 nL, about 9.5 nL,about 10 nL, about 11 nL, about 12 nL, about 13 nL, about 14 nL, about15 nL, about 16 nL, about 17 nL, about 18 nL, about 19 nL, about 20 nL,about 25 nL, about 30 nL, about 35 nL, about 40 nL, about 45 nL, orabout 50 nL.

A digital readout assay, e.g., digital analysis, can be used to detectand quantify RNA or DNA in a sample by partitioning the sample andreaction components and then amplifying the reactions with the primersand as described herein with at least two sets of amplification cycles.Generally, the process of digital analysis involves determining for eachpartition of a sample whether the partition is positive or negative forthe presence of the label or labels to be detected. For quantificationthe partitions are examined for the presence or absence of a detectablesignal in each partition. A partition is “positive” for the presence ofthe antigen if a signal is detected in the partition. A partition is“negative” if no signal detected in the partition.

In some embodiments, a detector that is capable of detecting a signal ormultiple signals is used to analyze each partition for the presence orabsence of signal. For example, in some embodiments a two-color reader(fluorescence detector) is used. The fraction of positive-countedpartitions can enable the determination of absolute concentrations forthe target DNA or RNA to be measured.

Once a binary “yes-no” result has been determined for each of thepartitions of the sample, the data for the partitions is analyzed usingan algorithm based on Poisson statistics to quantify the amount oftarget in the sample. Statistical methods for quantifying theconcentration or amount of target are described, for example, in WO2010/036352, which is incorporated by reference herein in its entirety.

DNA polymerases useful in the present invention can be any polymerasecapable of replicating a DNA molecule. Exemplary DNA polymerases arethermostable polymerases, which are especially useful in PCR.Thermostable polymerases are isolated from a wide variety ofthermophilic bacteria, such as Thermus aquaticus (Taq), Thermusbrockianus (Tbr), Thermus flavus (Tfl), Thermus ruber (Tru), Thermusthermophilus (Tth), Thermococcus litoralis (Tli) and other species ofthe Thermococcus genus, Thermoplasma acidophilum (Tac), Thermotoganeapolitana (Tne), Thermotoga maritima (Tma), and other species of theThermotoga genus, Pyrococcus furiosus (Pfu), Pyrococcus woesei (Pwo) andother species of the Pyrococcus genus, Bacillus sterothermophilus (Bst),Sulfolobus acidocaldarius (Sac) Sulfolobus solfataricus (Sso),Pyrodictium occultum (Poc), Pyrodictium abyssi (Pab), andMethanobacterium thermautotrophicum (Mth), and mutants, variants orderivatives thereof.

In some embodiments, the polymerase enzyme is a hybrid polymerasecomprising a polymerase domain and a DNA binding domain. Such hybridpolymerases are known to show an increased processivity. See e.g., U.S.Patent Application Publication Nos. 2006/005174; 2004/0219558;2004/0214194; 2004/0191825; 2004/0081963; 2004/0002076; 2003/0162173;2003/0148330; 2003/0138830 and U.S. Pat. Nos. 6,627,424 and 7,445,898,each of which is hereby incorporated by reference in its entirety forall purposes and in particular for all teachings related to polymerases,hybrid/chimeric polymerases, as well as all methods for making and usingsuch polymerases. In one aspect, the hybrid polymerases lack 3′-5′exonuclease activity. In one embodiment, such hybrid polymerasescomprise a double point mutation in the polymerase domain that providesthis exonuclease deficiency. In a specific embodiment, hybridpolymerases can comprise the double point mutation D141A/E143A in thepolymerase domain.

In some embodiments, the binding domain of hybrid polymerases is from athermostable organism and provides enhanced primer annealing at highertemperatures, e.g., temperatures above 45° C. For example, Sso7d andSac7d are small (about 7 kd MW), basic chromosomal proteins from thehyperthermophilic archaeabacteria Sulfolobus solfataricus and S.acidocaldarius, respectively (see, e.g., Choli et al., Biochimica etBiophysica Acta 950:193-203, 1988; Baumann et al., Structural Biol.1:808-819, 1994; and Gao et al, Nature Struc. Biol. 5:782-786, 1998).These proteins bind DNA in a sequence-independent manner and when bound,increase the Tm of DNA by up to 40° C. under some conditions (McAfee etal., Biochemistry 34:10063-10077, 1995). These proteins and theirhomologs are often used as the sequence-non-specific DNA binding domainin improved polymerase fusion proteins. Sso7d, Sac7d, Sac7e and relatedsequences (referred to herein as “Sso7 sequences” or “Sso7 domains”) areknown in the art (see, e.g., accession numbers (P39476 (Sso7d); P13123(Sac7d); and P13125 (Sac7e)). These sequences typically have at least75% or greater, of 80%, 85%, 90%, or 95% or greater, amino acid sequenceidentity. For example, an Sso7 protein typically has at least 75%identity to an Sso7d sequence.

In further embodiments, hybrid polymerases of use are described forexample in U.S. Patent Application Publication Nos. 2006/005174;2004/0219558; 2004/0214194; 2004/0191825; 2004/0081963; 2004/0002076;2003/0162173; 2003/0148330; 2003/0138830; PCT Publication No. WO2012/138417; and U.S. Pat. Nos. 6,627,424 and 7,445,898, each of whichis hereby incorporated by reference in its entirety for all purposes andin particular for all teachings related to polymerases, hybrid/chimericpolymerases, as well as all methods for making and using suchpolymerases. Examples of hybrid polymerase proteins and methods ofgenerating hybrid proteins are also disclosed in WO2004011605, which ishereby incorporated by reference in its entirety for all purposes, andin particular for all teachings related to generating hybrid proteins.

Many of the steps (e.g., reverse transcription, amplification, etc.)described above can be performed using routine conditions used in thefield of recombinant genetics. Basic texts disclosing the generalmethods of use in this invention include Sambrook and Russell, MolecularCloning, A Laboratory Manual (3rd ed. 2001); Kriegler, Gene Transfer andExpression: A Laboratory Manual (1990); and Current Protocols inMolecular Biology (Ausubel et al., eds., 1994-1999).

EXAMPLE

FIGS. 6-8 provide example schemes for various aspects. FIG. 6illustrates hypothetical use of polyadenylase to a target RNA (“PolyAreaction”). The poly adenylated RNA is subsequently reverse transcribedwith an RT primer. The RT primer includes a non-T nucleotide (in thisexample a G) in the middle of the homopolymeric polyT sequence thatcomplements the added polyA sequence on the RNA, thereby producing acDNA with a 5′ polyT sequence comprising the non-T nucleotide. A PCRreaction is subsequently performed (optionally a split cycle asdescribed herein) using a primer comprising a portion of the polyTsequence and having the intervening non-T nucleotide (for example, thePCR primer 2 underlined sequence). Both PCR primers uses include a 5′tail sequence shown in bold.

FIG. 7 illustrates a second hypothetical example in which a tailedprimer is used. The RT reaction section of the figure shows a RT primercomprising a 3′ section complementary to the target RNA and anunderlined 5′ tail that is not complementary to the target RNA.Following reverse transcription the shown “cDNA product” is produced.The cDNA product is then amplified in a PCR reaction (e.g., a splitcycle amplification as described herein) using Primer 1, which has abolded 5′ non-complementary tail and a 3′ section (normal font)complementary to the cDNA product, and Primer 2, which comprises a 5′non-complementary tail, a middle segment (underlined) that contains atleast a portion of the 5′ tail of the cDNA as introduced by the RTprimer 5′ tail, and a 3′ region that is complementary to the resultingdouble-stranded cDNA.

FIG. 8 illustrates a hypothetical example involving introduction of ahomopolymeric sequence using terminal transferase. In the RT reaction,the target RNA is reverse transcribed with a specific selective RTprimer that is complementary to the target RNA. While a 5′ tail is notused in this example, one could use such a tail. Subsequently, the cDNAis used in a terminal transferase to add a homopolymeric sequence (asshown, polyT) to the 5′ end of the cDNA. The cDNA product is thenamplified in a PCR reaction (e.g., a split cycle amplification asdescribed herein) using Primer 1 having a bolded 5′ non-complementarytail and a 3′ region complementary to the cRNA (normal font) and Primer2 in-frame with the cDNA strand and comprising a 5′ non-complementarytail section, an underlined sequence comprising at least a portion ofthe homopolymeric sequence, and a 3′ region.

In a working example, Synthetic DNA (22 nucleotides plus homopolymersequence of 15 Adenosine nucleotides) and primers containing tailed 5′regions as described (Integrated DNA Technologies, Coralville, Iowa)were added to EvaGreen ddPCR Supermix Cat #186-4034 (Bio-RadLaboratories, Hercules, Calif.). The reaction mix was made with allcomponents added to one 50 μL reaction and 20 μL was pipetted into twoseparate wells for droplet generation using the QX200 ddPCR system(Bio-Rad Laboratories, Hercules, Calif.). The droplet reactions fromthese wells were added each to a separate plate where one was thermalcycled using the standard thermal cycling protocol 95° C. for 5 minutes,40 cycles of: 95° C. for 30 seconds and 50° C. for 1 minute, 4° C. for 5minutes, 90° C. for 5 minutes, 4° C. hold and the other was thermalcycled using the split cycle thermal cycling protocol 95° C. for 5minutes, 10 cycles of: 95° C. for 30 seconds and 40° C. for 1 minute, 30cycles of: 95° C. for 30 seconds and 50° C. for 1 minute, 4° C. for 5minutes, 90° C. for 5 minutes, 4° C. hold. The table shows that thecopies per microliter concentrations were 25% higher in the split cycledwell compared to the standard one temperature thermal cycling protocol.Further repeated experiments showed more efficient amplification wasconsistently seen as fewer mid-amplitude positives and quantification of25-50% higher copies per microliter when the split cycle thermal cyclingprotocol was used in combination with assays having 5′ tail regions(shown in FIG. 1).

Thermalcycling Protocol 40 cycles: 50° C. 10 cycles: 40° C. 30 cycles:50° C. Copies/μL 67.8 89.5

An alignment is provided below. The alignment of the human microRNA let7 family sequences with the sequences that differ from the let-7a-5phighlighted in bold and underlined (SEQ ID NOs:4-12, respectively).

hsa-let-7a-5p A A C T A T A C A A C C T A C T A C C T C A hsa-let-7b-5pA A C  C  A  C  A C A A C C T A C T A C C T C A hsa-let-7c-5p A A C  C A T A C A A C C T A C T A C C T C A hsa-let-7d-5p A A C T A T  G C A A C C T A C T A C C T C  T hsa-let-7e-5p A A C T A T A C A A C C T C  C T A C C T C A hsa-let-7f-5p A A C T A T A C A A  T C T A C T A C C T C A hsa-let-7g-5p A A C T  G  T A C A A  A C T A C T A C C T C A hsa-let-7i-5p A A C  A   G   C  A C A A  A C T A C T A C C T C A hsa-miR-98-5p A A C  A  A T A C A A C  T T A C T A C C T C A

Discrimination of each of these 9 highly similar microRNAs was achievedusing a combination of the split cycle thermal cycling with the reversetranscription primer having the homopolymer region interspersed with asingle, non-homopolymer nucleotide, the discriminating nucleotide placedwithin 1, 2 ,3, or 4 of the 3′ end of the primer(s), and in some casesan added mismatch to further destabilize binding of the primer to theincorrect target. The results of the assay as shown below:

The percent RNA amplified and detected for each of the family membermicroRNAs are shown above for each of the 9 different assays. The assayname is shown along the left vertical axis and the synthetic microRNAtemplate added is shown along the top horizontal axis. The chart showsthe percent of the sample that was amplified and measured.

Synthetic microRNA for all 9 hsa-miR-let-7 (22 nucleotides each), thereverse transcription primer and 5′ tailed assay primers were purchased(Integrated DNA Technologies, Coralville, Iowa). Synthetic microRNA hada homopolymer region of poly adenosine added to the microRNA using apoly adenylase enzyme (New England Biolabs, Ipswich, Mass.). This polyadenylated synthetic RNA was then added to a gene specific reversetranscription reaction using iScript Select cDNA synthesis kit cat#170-8896 (Bio-Rad Laboratories, Hercules, Calif.) and the reversetranscription primer with a homopolymer having partial complementarityto the poly adenylated region and synthetic microRNA as shown in FIG.3A. The cDNA generated in the reverse transcription reaction was addeddirectly to EvaGreen ddPCR Supermix Cat #186-4034 (Bio-Rad Laboratories,Hercules, Calif.) with the 5′ tailed primers described in FIGS. 3B & 3C.Cross reactivity or off-target amplification for the 9 nearly identicalmicroRNA targets from the let-7 family shown in panel A was assessed bytesting each discriminating assay individually with each of the 9targets. Percent cross reactivity was calculated by setting theconcentration of the target detected with a non-discriminating assay(FIG. 1) as 100%. Cross reactivity was calculated by dividing theconcentration of the target that was amplified when each of the 9targets were added individually to each assay by the concentration for100% target amplification. All assays tested were able to discriminatewith less than 4% cross reactivity for all 9 targets (less than 1% crossreactivity for with the exception of three 1.6, 1.7 and 3.5%) (B). SomemicroRNA methods need to sacrifice efficiency in order to obtain lowcross reactivity but with the methods described here we demonstrate thatwe are able to obtain a high degree of specificity (low crossreactivity) with 100% efficiency.

All documents (for example, patents, patent applications, books, journalarticles, or other publications) cited herein are incorporated byreference in their entirety and for all purposes, to the same extent asif each individual document was specifically and individually indicatedto be incorporated by reference in its entirety for all purposes. To theextent such documents incorporated by reference contradict thedisclosure contained in the specification, the specification is intendedto supersede and/or take precedence over any contradictory material.

Many modifications and variations of this invention can be made withoutdeparting from its spirit and scope, as will be apparent to thoseskilled in the art. The specific embodiments described herein areoffered by way of example only and are not meant to be limiting in anyway.

What is claimed is:
 1. A method of generating and amplifying cDNA, themethod comprising: (a) reverse transcribing a target RNA comprising anon-polyA region and a polyA tail by: (i) hybridizing a reversetranscription (RT) primer to the target RNA, wherein the RT primercomprises from 5′ to 3′:X-(T)_(m)-Y-(T)_(n)-Z wherein X is an optional 5′ tail nucleotidesequence of 1-10 nucleotides that is not complementary to the targetRNA; T is thymine; Y is a single nucleotide that is C, G, or A; Z is anoptional sequence of 1-10 nucleotides that is complementary to a portionof the non-polyA region adjacent to the polyA tail; m is 1-20; and n is1-20; and (ii) reverse transcribing the target RNA by extending the RTprimer with a RNA-dependent polymerase to generate a cDNA incorporatingthe RT primer; and (b) amplifying the cDNA by: (i) hybridizing anamplification primer comprising a sequence complementary to the-(T)_(m)-Y-(T)_(n), and (ii) extending the amplification primer with aDNA-dependent polymerase.
 2. The method of claim 1, wherein m is 1-4 or2-4.
 3. The method of claim 1, wherein n is 1-4 or 2-4.
 4. The method ofclaim 1, wherein both m and n are independently 1-4 or 2-4.
 5. Themethod of claim 1, wherein the RT primer comprises Z and wherein theamplification primer comprises one or more 3′ nucleotides that arecomplementary to Z adjacent to the sequence complementary to the-(T)_(m)-Y-(T)_(n).
 6. The method of claim 5, wherein Z is 1-5 or 2-5nucleotides in length.
 7. The method of claim 1, wherein theRNA-dependent polymerase is a reverse transcriptase selected from thegroup consisting of murine leukemia virus (MLV) reverse transcriptase,Avian Myeloblastosis Virus (AMV) reverse transcriptase, RespiratorySyncytial Virus (RSV) reverse transcriptase, Equine Infectious AnemiaVirus (EIAV) reverse transcriptase, Rous-associated Virus-2 (RAV2)reverse transcriptase, SUPERSCRIPT II reverse transcriptase, SUPERSCRIPTI reverse transcriptase, THERMOSCRIPT reverse transcriptase, and MMLVRNase H⁻ reverse transcriptase.
 8. The method of claim 1, wherein theamplifying comprises a polymerase chain reaction.
 9. The method of claim8, wherein the amplifying comprises a digital PCR reaction.
 10. Themethod of claim 8, wherein the amplifying comprises at least twodifferent sets of amplification cycles, wherein the second set ofamplification cycles has an annealing temperature that is higher than anannealing temperature of the first set of amplification cycles.
 11. Themethod of claim 10, wherein the second set of amplification cycles hasan annealing temperature that is at least 5° C. higher than theannealing temperature of the first set of amplification cycles.
 12. Themethod of claim 10, wherein the first set of amplification cycles has afewer number of cycles than the second set of amplification cycles. 13.The method of claim 12, wherein the first set of amplification cycleshas between 5-15 cycles.
 14. The method of claim 12, wherein the secondset of amplification cycles has between 15-45 cycles.
 15. The method ofclaim 12, wherein the first set of amplification cycles has between 5-15cycles and the second set of amplification cycles has between 15-45cycles.
 16. The method of claim 1, wherein the amplifying comprises theuse of an intercalating agent that generates a signal when intercalatedin double stranded DNA.
 17. The method of claim 1, wherein the targetRNA is a microRNA (miRNA), small nucleoplasmic RNA (snRNA), smallnucleolar RNA (snoRNA), Piwi-interacting RNA (piRNA), or long noncodingRNA (lncRNA).